The effects of various antibiotics on the biosynthesis of lipid-linked oligosaccharides is being studied in cell-free extracts of pig aorta. We have studied three antibiotics, tunicamycin, bacitracin and amphomycin, which inhibit various steps in these reactions. Tunicamycin was previously shown by several laboratories to inhibit the formation of G1cNAc-pyrophosphoryl-dolichol, the first glycolipid formed on this pathway. We examined the effect of this antibiotic on the solubilized and partially purified GlcNAc transferase. The inhibition by tunicamycin could not be overcome by increasing the concentrations of any of the substrates, i.e., UDP-G1cNAc, dolichyl phosphate or Mn ions indicating that it was a non-competitive inhibitor. Tunicamycin also inhibited the reverse reaction. Another antibiotic, bacitracin was found to inhibit the formation of both G1cNAc-pyrophosphoryl-dolichol and mannosyl-phosphoryl-dolichol in both the particulate and solubilized enzyme. It also inhibited mannose transfer from mannosyl-phosphoryl-dolichol and from lipid-linked oligosaccharides. Amphomycin is another antibiotic which blocks the formation of both GlcNAc-pyrophosphoryl-dolichol and mannosyl-phsophoryl-dolichol. However, in the presence of amphomycin, mannose was still transferred from GDP-mannose to lipid-linked oligosaccharides indicating a direct mannose transfer. But in this case, the radioactivity was in one oligosaccharide which appeared to be a heptasaccharide rather than in a number of different sized oligosaccharides.